Compositions and methods of treatment for inflammatory diseases

ABSTRACT

Inflammatory bowel diseases are represented by two idiopathic disorders, which include ulcerative colitis and Crohn&#39;s disease. Ulcerative colitis is restricted to the colon and involves uncertain and inflammation of the lining (mucosa) of the large intestine. Crohn&#39;s disease, on the other hand, can involve the mucosa of the small and/or large intestine and may involve deeper layers of the bowel wall. The present invention is a combination of 5-aminosalicylic acid and one or more antioxidants (e.g., N-acetylcysteine) for treating such inflammatory bowel diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/402,378, filed on Mar. 11, 2009, by Richard F. Harty, and entitled“COMPOSITIONS AND METHODS OF TREATMENT FOR INFLAMMATORY DISEASES,” nowU.S. Pat. No. 8,119,618, issued on Feb. 21, 2012, which is acontinuation of U.S. application Ser. No. 11/347,113, filed on Feb. 2,2006, by Richard F. Harty, and entitled “COMPOSITIONS AND METHODS OFTREATMENT FOR INFLAMMATORY DISEASES,” now U.S. Pat. No. 7,691,832,issued on Apr. 6, 2010, which is a continuation of U.S. application Ser.No. 11/023,812, filed on Dec. 28, 2004, now U.S. Pat. No. 7,417,037,issued on Aug. 26, 2008, by Richard F. Harty, and entitled “COMPOSITIONSAND METHODS OF TREATMENT FOR INFLAMMATORY DISEASES,” which in turnclaims the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalApplication Ser. No. 60/537,766, filed Jan. 20, 2004, the entiredisclosure of all of which is hereby expressly incorporated by referenceherein.

BACKGROUND

Inflammatory bowel diseases (IBDs) including ulcerative colitis andCrohn's disease, are complex diseases that are thought to result fromover activation of the immune system directed at luminal antigens of thegastrointestinal tract (12). In the early 1940's it was observed thatsulfasalazine, formed by the chemical union of the antibioticsulfapyridine and 5-aminosalicylic acid (5-ASA; also referred to asmesalamine) by an azo bond, had a beneficial effect in patients withcolitis (29). Subsequent clinical studies over the next two decadesestablished that sulfasalazine had efficacy in the treatment ofinflammatory bowel disease (30, 31). Additional studies were directed todetermine the chemical kinetics of sulfasalazine when administeredorally and to determine mechanisms of action (32-34). Approximate 75% ofsulfasalazine reaches the colon unchanged. Within the colon the azo bondis split by bacterial enzyme action into metabolites, 5-ASA andsulfapyridine. Following azo bond reduction, most of the sulfapyridineis absorbed from the colon whereas only 20% of 5-ASA is absorbed. Themajority of 5-ASA remains in the colon and is recovered in the fecesprimarily as free 5-ASA.

Postulated mechanisms for the presumed beneficial action ofsulfasalazine in the treatment of colitis initially included inhibitionof prostaglandin synthesis and inhibition of the lipoxygenase pathway ininflammatory cells such as neutrophils (35,36). Ensuing investigationshave established additional therapeutic mechanisms whereby 5-ASApromotes healing and reduces inflammation in IBD (2, 37, 38). Theseinclude: free radical scavengers, inhibit T-cell proliferation, inhibitpresentation of antigen to T-cells, inhibit adhesion of macrophages andgranulocytes, decrease production of interleukins (ILs) and downregulation of the transcription factor, NF-kB, activity. Despite theutility of sulfasalazine for patients with inflammatory bowel disease,experience has shown that up to one third of patients cannot toleratethis medication and manifest one or more side effects of variableseverity. These side effects are related directly to systemic absorptionof sulfapyridine. Because of sulfasalazine-related side effects,investigators have examined 5-ASA as a single agent for the treatment ofinflammatory bowel disease. There have been several formulations of5-ASA designed to inhibit proximal intestinal absorption and delivery ofthis compound to areas of active inflammation (39). Several formulationsof 5-ASA have been studied and those currently most popular includecoated forms of 5-ASA that are released in a pH-sensitive manner to thedistal ileum and colon. Examples of such agents include Asacol™ (Proctorand Gamble) and Pentasa™ (Shire US inc.). Furthermore, 5-ASA preparationfor rectal delivery has included the development of suppositories andenemas containing 5-ASA as the active agent. Examples include Rowasa™rectal suspension enema (Solvay Pharmaceuticals) and 5-ASA suppositoriessuch as Canasa™ (Axcan Scandiapharm). Thus, a number of oral or rectallydelivered 5-ASA agents are presently available for the treatment of mildto moderate inflammatory bowel disease.

Recent investigations into the etiological triad of genetic:environmental: immune factors have expanded our knowledge of theseindividual components and their potential interactions. Pathogeneticmodels of IBD envision initiating events, possible microbiologicals,arising from the gut lumen that converts immune tolerance to a sustainedhyperactive state with elaboration and amplification of cellular andhumoral mediators. Immunocyte derived injurious and proinflammatorysubstances cause tissue injury and destruction. These substances includeprostaglandins, reactive oxygen metabolites, nitric oxide, leukotrienes,proteases and matrix metalloproteinases (3). The role of reactive oxygenspecies (ROS) and nitric oxide (NO) have been examined in experimentalmodels of IBD (4, 5). Pharmacological and genetic manipulation of oxygenfree radical and NO generation have been shown to ameliorateexperimental colitis induced by luminal administered trinitrobenzenesulfonic acid (TNBS) and dextran sulfate sodium (DSS) (5-10).

Although several experimental strategies have been employed that suggestthe importance of enhanced production of superoxide and nitric oxide inthe pathogenesis of IBD, inconsistent results have the issue unresolved.For example, the beneficial effect of superoxide dismutase (SOD)treatment in experimental models of colitis has been reported while SODtreatment in humans with IBD has shown limited benefit (40). Similarly,the inhibitors of inducible nitric oxide synthase (iNOS) have yieldedmixed results in various experimental models of IBD (6-8, 27). Theantioxidants N-acetylcysteine (NAC) and phenyl N-tert-butylnitrone (PBN)when used alone have been shown to be effective in protection againstTNBS-induced colitis in rat (9) and DSS-induced colitis in mice, (10)respectfully. Furthermore, recent studies suggest a dominant role ofiNOS-derived NO in a murine model of colitis (5). Antioxidant therapyhas also been shown to suppress colonic iNOS activity and to decreasecolonic NF-κB DNA-binding activity in experimental animals (10). Nuclearfactor-κB, NF-κB is a family of transcription factors known to regulatea variety of genes controlling the inflammatory process and regulatingprogrammed cell death (41).

Thus, there exists extensive experimental support for the notion thatreactive oxygen molecules and nitric oxide may contribute to thepathogenesis of mucosal injury in inflammatory bowel disease.Furthermore, experimental evidence also provides support for the conceptthat inhibition of nitric oxide species and NO generation exertfavorable effects on mucosal healing and the inflammatory process inseveral well-defined models of chemically induced colitis. However,there continues to be a need in the field for a more effective treatmentof inflammatory bowel diseases and other conditions related toinflammation. It is to this need that the present invention is directed.

SUMMARY OF THE INVENTION

Disclosed herein are methods of treating an inflammatory bowel diseasecomprising administering a composition comprising 5-aminosalicylic acidand N-acetylcysteine to a mammal or contacting a colonic cell of themammal with the said composition. Also disclosed are methods of reducingcytokine gene expression or reducing myeloperoxidase activity in colonictissue of a mammal, the method comprising administering a compositioncomprising 5-aminosalicylic acid and N-acetylcysteine to a mammal orcontacting a colonic cell of the mammal with the said composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Experimental Design: Colitis was induced at day 0 by rectaladministration of TNBS. Three days after TNBS dosing rats received dailyintraluminal therapy per rectum for either 5 or 8 days. Four treatmentprotocols were employed and included: (A) 5-ASA (100 mg/kg), (B) NAC (40mM), (C) a combination of 5-ASA (100 mg/kg) and NAC (40 mM), and (D) asaline control.

FIG. 2: Macroscopic grading of inflammation and injury 8 days afterintracolonic administration of TNBS. Macroscopic grading (0-6) 8 daysafter TNBS revealed moderately severe inflammation and ulceration: score4.5±0.5. Therapy with the NAC/5-ASA combination for 5 d was the onlyintervention that significantly reduced macroscopic injury: score2.6±0.7. These results are representative of 4 different experimentsdone at different times with 5 animals per group per each experiment. *denotes p<0.05 vs TNBS.

FIG. 3: Macroscopic injury score 11 days after TNBS and 8 days oftreatment with 5-ASA alone, NAC alone and the NAC and 5-ASA combination.

FIG. 4: Microscopic grading of inflammation and injury 8 days afterintracolonic administration of TNBS. Microscopically, TNBS affectedsignificantly each parameter of injury and inflammation as reflected bya cumulative microscopic injury score (0-15) of 9.4±1.0 p<0.001. TheNAC/5-ASA combination significantly reduced the cumulative microscopicinjury: score 5.0±1.2 p<0.001. Single agent therapy with either NACalone or 5-ASA alone did not significantly decrease microscopic injury:NAC 9.0±1.8, and 5-ASA 9.0±0.9. These results are representative of 4different experiments done at different times with 5 animals per groupper experiment. * denotes p<0.05 vs TNBS.

FIG. 5: Combination therapy with the NAC/5-ASA combination for 8 daysreduced significantly the aggregate microscopic injury score by 75%.Therapy was initiated 3 days after induction of colitis by TNBS.Furthermore, single agent therapy with 5-ASA alone and NAC alone alsosignificantly decreased histological measures of injury by 46 and 53%,respectively. The degree of histologic healing with the NAC/5-ASAcombination was significantly greater than that observed with either5-ASA or NAC alone.

FIG. 6: Myeloperoxidase activity in colon tissue 11 days after TNBS and8 days of treatment with 5-ASA alone, NAC alone and the NAC plus 5-ASAcombination. Results represent the mean±SEM of 4-6 rats per condition.

DESCRIPTION OF THE INVENTION

The present invention contemplates use of 5-ASA (mesalamine) plus anantioxidant either together as separate molecular entities or whencoupled chemically, to provide an enhanced therapeutic or prophylacticeffect against inflammatory bowel diseases in mammals. The combinationof mesalamine plus an antioxidant in a mammal model of colitis promoteshealing and reduces inflammation to a significantly greater degree thaneither agent when used alone. The present invention provides an improvedanti-inflammatory effectiveness of 5-ASA for use in the treatment ofinflammatory bowel disease by the addition of an antioxidant. Use of thepresently claimed compositions result in a reduction in the need foradditional anti-inflammatory agents, such as prednisone and Imuran inthe treatment of inflammatory disease (thereby providing a significantcost benefit and reduction in drug-induced toxicity.). Furthermore, thepresent invention can serve as a model for additional pharmacologicalapproaches to other inflammatory and, possibly, neoplastic conditionswithin the gastrointestinal tract.

As shown below experiments were performed to examine the ability of anantioxidant (NAC) and 5-ASA, when administered separately or incombination to the distal colon, to affect mucosal healing and repairfollowing colitis chemically-induced by TNBS or DSS.

Materials and Methods

Experimental Animals

Male Sprague Dawley rats (200-250 grams) were housed in cages containingcontact bedding. Rats were deprived of food for 24 hrs prior to theinduction of colitis, but were allowed free access to water throughoutthe experiment. Institutional approval for experimental protocols wasprovided by the research and animal care committees of the researchservices at the Oklahoma City Veterans Administration Medical Center.

Induction of Colitis by TNBS Treatment

Following a 24 hr fast, rats were lightly anesthetized using isofluranefollowed by insertion of a polyethylene catheter into the anus. The tipof the catheter was advanced 8 cm proximal from the anus and a singledose of TNBS (Sigma, St. Louis, Mo.), dissolved in 50% ethanol, wasinjected through the cannula (15 mg dissolved in a 0.6 ml volume of 50%ethanol) to induce colitis. Following the administration of TNBS theanimals were maintained in a head-down position for approximately 60seconds to prevent leakage of the infusate. After 72 hrs, rats dosedinitially with TNBS were randomly assigned to one of four treatmentgroups to receive daily intracolonic therapy in a manner similar to thatused for TNBS administration. The groups were: A.) 5-aminosalicyclicacid (5-ASA) (100 mg/kg) B.) N-acetylcysteine (NAC) (40 mM) plus 5-ASA(100 mg/kg) C.) NAC (40 mM), or D.) saline. An additional group of rats(Group E) served as control and received saline per rectum on day 0 andat subsequent intervals that corresponded to treatment protocols. Ratswere treated for either 5 or 8 days and then sacrificed by cervicaldislocation. FIG. 1 illustrates the design of these experiments.

Induction of Colitis by DSS Treatment

Colitis was induced by oral administration of a 4% solution of DSS(dextran sulfate sodium) in drinking water for 4 days. Experimentalgroups were randomized to include group 1 control, group 2-5 receivedDSS in drinking water and groups 3-5 were further randomized to receivedaily intracolonic treatment with 5-ASA (100 mg/kg) plus NAC (40 mM),group 3; 5-ASA alone, group 4; and NAC constituted group 5. Animalsreceived treatment for 4 days. Macroscopic indices of colonic injurywere scored and tabulated. Histological features that were examinedincluded epithelial damage and mucosal ulceration. Additional measuresincluded determination of serum C-reactive protein (CRP) levels andcytokine gene expression in colonic tissues performed by ribonucleaseprotection assay (RPA).

Macroscopic Assessment of Colitis

Upon sacrifice, the distal 8 cm of the colon was removed, opened by alongitudinal incision and rinsed with phosphate buffered saline toremove fecal material. Macroscopic assessment of colitis was carried outby an independent observer who was unaware of the treatment groups. Thecriterial and scale of grading are listed in Table 1. Inflammation waspresent if the mucosa was erythematous. Ulceration of the mucosa wasdefined as a distinct break or interruption of the mucosa. Oncemacroscopic damage was assessed full thickness colonic tissue sampleswere taken from the inflamed areas and either processed for histology orsnap frozen in liquid nitrogen for subsequent cytokine measurement anddetermination of myeloperoxidase (MPO) activity.

TABLE 1 Macroscopic Scoring of colitis Macroscopic Injury Score Normal 0One area of inflammation 1 No ulcer No inflammation 2 One ulcer One areaof inflammation 3 One or two ulcers One area of inflammation 4 More than2 ulcers Two areas of inflammation 5 More than 2 ulcers Ulceration >2 cm6

Histological Assessment of Colitis

Colonic tissue samples taken from the initial segment were immersed in10% phosphate buffered formalin and subsequently embedded in paraffin.Sections of 3 micron thickness were cut and stained with hematoxylin andeosin. The slides were then evaluated by a pathologist for epithelialdamage, architectural changes, mononuclear infiltration,polymorphonuclear leukocyte (PMN) infiltration and ulceration. Theindividual microscopic features of colitis were graded according tocriteria noted in Table 2. In addition to scoring individual features ofcolitis, an aggregate score of colitis was tabulated by adding togetherindividual scores, thus, providing a global assessment of colitis.

TABLE 2 Histological Scoring of Colitis Epithilial Damage 0 Normal 1Focal Mucosal injury 2 Extensive mucosal injury Architectural Damage 0Normal 1 Moderately disturbed 2 Severely disturbed MononuclearInfiltration 0 Normal 1 Moderate increase 2 Severe increase PMNInfiltration 0 Normal 1 Present In surface epithelium 2 Cryptitis 3Crypt abscesses Ulcerations 0 None 1  1%-33% ulcerated 2 34%-66%ulcerated 3 67% + ulcerated

Myeloperoxidase Activity

Myeloperoxidase activity was used as an indirect measure of the severityof colonic inflammation by PMNs. Whole thickness tissues taken followingmacroscopic assessment were weighed (100 mg) and immediately snap frozenin liquid nitrogen for storage at −80.degree. C. The tissues were thenremoved from storage at −80° C. and allowed to thaw on ice. Once thawed,1 ml of hexadecyltrimethylammonium bromide (HTAB) (Sigma, St. Louis,Mo.) containing 50 mM KH₂PO₄ (Sigma, St. Louis, Mo.) and 0.1M Na₂HPO₄(Sigma, St. Louis, Mo.) was added per 100 mg tissue for homogenization.Homogenates then underwent a series of four freeze/thaw cycles beforefinally being centrifuged at 12,000×g for 10 minutes at 4° C. Thesupernatant was collected for measurement of MPO activity. Horseradishperoxidase (Sigma, St. Louis, Mo.) was used as a standard; stocksolution of 0.5 mg/ml. Tetramethylbenzidine (TMB) (Sigma, St. Louis,Mo.) was used as the substrate for carrying out the reaction. At thetime of assay 25 μl of standard and sample were added to appropriatelylabeled tubes. TMB was added at a volume of 250 μl to initiate thereaction and 0.1M H₂SO₄ (250 μl) was added after 10 minutes to terminatethe reaction. The absorbance changes were read at 450 nm and recorded.Results were expressed as ng/ml/g of tissue.

Ribonuclease Protection Assay (RPA)

Following the manufacturer's protocol, 1 ml of TRI Reagent (Sigma, St.Louis, Mo.) is used per 100 mg of tissue. The frozen tissue is placedinto the TRI Reagent and immediately homogenized using a tissuemacerator. The homogenates are transferred to microcentrifuge tubes and0.2 ml of chloroform is added per ml of TRI Reagent used. The samplesare shaken thoroughly and allowed to sit at room temp for 3 min. Sampleswere then centrifuged at 12000×g for 15 min at 40° C. Aftercentrifugation, the upper aqueous phase was transferred to a new tubeand 9.5 ml of 2-propanol is added to precipitate the RNA. After sittingat room temp for 10 min. the samples are centrifuged at 12000×g for 15min. The supernatants are discarded and the pellets are washed in 70%ethanol followed by centrifugation at 12000×g for 5 min. The ethanol wasremoved and the pellets are dissolved in 50 μl of DEPC treated water persample. Quantitation is performed by spectrophotometry at 260 nm and 280nm.

RPA analysis was performed with RiboQuant™ multi-probe RNase protectionassay system (BD Biosciences-Pharmingen, San Diego, Calif.). To 20 μg oftotal RNA in 8 μl of hybridization buffer and 2 μl of ³²P-labeled invitro transcribed RNA probes were added. The probes were transcribedfrom the multiprobe template set rCK-1 which as a panel of probesagainst IL-1a, IL-1b, IL-3, IL-5, IL-10, IL-2, TNFα, TNBβ, GAPDH genes.Probes were prepared according to the manufactures protocol. The RNAsamples containing labeled probes were incubated briefly at 90° C.,slowly cooled to 56° C. and hybridized overnight at that temperature.The samples were then slowly cooled to 30° C. Thereafter, a mixture ofRNases A and T1 was added to digest single-stranded RNA leaving thedouble-stranded RNA formed by the probes annealing to their cognatemRNAs intact. Following proteinase K digestion and phenol-chloroformextraction, the undigested double-stranded RNA was precipitated withammonium acetate and ethanol. After centrifugation at 14000×g for 20min., the pellets are washed with 90% ethanol and recentrifuged. Theethanol was removed and the pellets were dissolved in 5 μl of formamideloading dye. The double-stranded RNAs were resolved on a 5%polyacrylamide/urea gel run in TBE buffer. After electrophoresis, thegel was dried at 80° C. under vacuum and then exposed to aPhosphorImager screen overnight. Detection and quantitation of theresolved bands on the gel were performed on a Storm PhosphorImager™system (Amersham Biosciences, Piscataway, N.J.).

Statistical Significance

All values in the figures and text are expressed as means±standard errorof the mean (SEM). The statistical significance of any difference amonggroups was analyzed using Student's two-tailed t test for equal andunequal variance observations. P values of <0.05 were considered to bestatistically significant.

Results

Macroscopic Findings

8 Days After TNBS; 5-Day Treatment

The macroscopic injury observed at 8 days after rats received TNBS was4.5±0.5. This value indicated that the distal 8 cm of colon containedmore than 2 discrete ulcers in an area of inflammation (FIG. 2). Incontrast, combination therapy with the NAC plus 5-ASA combination actedsynergistically to cause a significant reduction in macroscopic injuryas reflected by an injury score of 2.6±0.7; p<0.05. Monotherapy witheither NAC or 5-ASA alone caused reduction in macroscopic injury(4.1±0.6 and 3.0±0.7, respectively) but these values did not achievesignificance when compared to TNBS alone.

11 Days After TNBS; 8-Day Treatment

Visual evidence of colitis 11 days after TNBS was scored at 4.1±0.2(FIG. 3). Animal treated with the NAC plus 5-ASA combination for 8 daysshowed no evidence of mucosal inflammation on ulceration. The colonicmucosa in these animals appeared normal. Monotherapy with either NAC or5-ASA alone showed mild inflammation and macroscopic injury scores were1.2±0.2 and 1.8±0.2 respectively.

Microscopic Findings

8 Days After TNBS; 5-Day Treatment

The cumulative or aggregate microscopic colitis injury score for ratsexamined 8 days after TNBS was 9.4±1.0 which represented moderatelysevere colitis (FIG. 4). Individual therapy for 5 days with either 5-ASAor NAC alone did not alter the aggregate injury score: 9.0±0.9 and9.0±1.8, respectively. Therapy with the NAC plus 5-ASA combination,however, caused significant reduction (−44%) in cumulative colitisinjury and resulted in a score of 5.0±1.2, P<0.02. Subset analysis ofindividual histological features, when compared to TNBS alone, indicatedthat therapy with the NAC plus 5-ASA combination acted synergisticallyto significantly reduce the degree of epithelial damage, the extent ofmucosal ulceration and the amount of mononuclear cell infiltration.

11 Days After TNBS; 8-Day Treatment

Eleven days post TNBS the indices of microscopic colitis were reducedslightly but not significantly from aggregate scores recorded at 8 daysafter TNBS: 7.0±0.9 vs 9.5±1.0; P>0.1. In contrast to the histologicalresults after 5 days of treatment each of the intraluminal therapies for8 days caused significant reduction in global measures of colitis (FIG.5). 5-ASA and NAC each when administered alone caused comparabledecrease in aggregate injury of 46 and 53%, respectively. Combinationtherapy with the NAC plus 5-ASA combination caused a 75% reduction incumulative colitis injury score: 1.8±0.5 vs 7.0±0.9 TNBS alone; P<0.001.Furthermore, comparisons between monotherapies with either 5-ASA or NACalone and combination therapy indicated that the NAC plus 5-ASAcombination acted synergistically to produce a significantly greaterdegree of healing than either NAC or 5-ASA alone.

Myeloperoxidase Activity

MPO activity in colonic tissue 11 days after TNBS treatment was elevatedgreater than 25 fold above values derived from saline treated rats (FIG.6). In contrast, the NAC plus 5-ASA combination treatment for 8 daysreduced MPO activity by 85% to 100.9±12.9 ng/g (P<0.004 vs TNBS690.2±101.5 ng/g). MPO activity was also reduced by both NAC and 5-ASAalone to levels that were 40% and 32% below TNBS values but thesedifferences did not achieve statistical significance.

Inflammatory Cytokine Expression

Cytokine gene expression in colonic tissues indicate that elevations ininterleukins (IL 1a, IL 1b, IL-4 and IL-6) and TNF a that were inducedby TNBS after 11 days were inhibited significantly by 8 day treatmentwith the NAC plus 5-ASA combination (Table 3). Treatments with eitherNAC or 5-ASA alone did not significantly suppress levels of cytokineexpression induced by TNBS.

TABLE 3 Cytokine gene expression in rat colon Relative O.D unitscytokine Treatment Group Mean ± SEM IL-1A Saline 0.54 ± 0.07 TNBS only1.97 ± 0.3  TNBS + 5ASA = NAC  0.70 ± 0.08** TNBS + 5ASA 1.33 ± 0.18TNBS + NAC 1.28 ± 0.16 IL-1B Saline 2.25 ± 0.37 TNBS only 11.77 ± 1.7 TNBS + 5ASA + NAC  6.03 ± 0.62** TNBS + 5ASA 11.49 ± 2.54  TNBS + NAC11.88 ± 1.0  IL-4 Saline 0.27 ± 0.06 TNBS only 1.76 ± 0.42 TNBS + 5ASA +NAC  0.82 ± 0.12** TNBS + 5ASA 1.40 ± 0.25 TNBS + NAC 1.20 ± 0.26 IL-6Saline 0.18 ± 0.03 TNBS only 1.21 ± 0.37 TNBS + 5ASA + NAC  0.46 ±0.06** TNBS + 5ASA 0.79 ± 0.22 TNBS + NAC 0.76 ± 0.14 TNF-α Saline 0.41± 0.06 TNBS only 1.51 ± 0.13 TNBS + 5ASA + NAC   0.7 ± 0.15** TNBS +5ASA 1.25 ± 0.12 TNBS + NAC  1.04 ± 0.017 **denotes a P value<0.05 vsTNBS alone Data represent the Mean ± SEM of 6-10 observations percondition

Results of the TNBS experiments of the present study indicate thatintraluminal administration of the NAC plus 5-ASA combination to thedistal colon of rats with TNBS colitis act synergistically to cause asignificant reduction in colonic inflammation and ulceration andacceleration of mucosal healing when compared to either agent usedalone. Furthermore, combination therapy with the NAC plus 5-ASAcombination caused significantly greater inhibition of myeloperoxidaseactivity and proinflammatory cytokine gene expression in colons of TNBStreated animals than either NAC or 5-ASA alone. Duration of treatmentwas a determinant in the effectiveness of antioxidant andanti-inflammatory agents on healing of chemically induced colitis. TheNAC plus 5-ASA combination was the only dosing regimen that resulted insignificant improvement in both macroscopic and microscopic measures ofcolitis after a five day treatment. In contrast, eight day treatmentwith NAC alone, 5-ASA alone or the NAC plus 5-ASA combination resultedin significant improvement in histological features of colitis.Treatment with the NAC plus 5-ASA combination, however, caused greaterimprovement in mucosal injury, inflammation and epithelial regenerationthan NAC or 5-ASA alone. Data derived from these studies indicate thatintraluminal therapy with the antioxidant NAC plus 5-ASA combination issuperior to either agent alone in the treatment of TNBS colitis and thatdual therapy has a synergistic effect in reducing inflammation andpromoting mucosal repair.

Colitis induced by intracolonic instillation of TNBS manifests many ofthe histological and clinical features of colonic inflammatory boweldisease (11). This model of colonic ulceration and transmuralinflammation of the mucosa (12) has been employed to study thepathogenesis of colonic inflammation and to investigate potentialtreatments of IBD. In this latter regard previous reports have shownthat both 5-ASA and NAC, when administered singly by intracolonic routeto rats, (9, 13) exerted an anti-inflammatory effect on TNBS colitis.Furthermore, the antioxidant NAC was observed to increase colonicglutathione stores which were associated with a reciprocal decrease inthe extent of mucosal injury (9). In support of these observations andthe role of ROS generation in TNBS colitis Loguercio et al reported thatglutathione supplementation improved oxidative damage in TNBS colitis(14).

Antioxidants, such as NAC, and 5-ASA possess the ability to scavengeoxygen free radicals, inhibit inducible NO formation and to downregulate nuclear factor κB (NF-κB) activity (9, 10, 15-17). Furthermore,antioxidants, such as phenyl N-tert-butylnitrone, and 5-ASA have beenshown to inhibit cytokine production, including tumor necrosis factorTNFα, and to retard adhesion module expression and B-cell mediatedantibody production (10, 18-20) in experimented models of colitis.Separately and together these agents can, thus, be envisioned tomoderate immunocyte (T cell) mediated cytokine elaboration, neutrophilgeneration of ROS and NO, prostaglandin release and to facilitate anenvironment for unopposed cellular and growth factor-mediated tissuerepair. These results show that treatment with the NAC plus 5-ASAcombination cause marked improvement in indices of colitis and,furthermore, demonstrate prominent features of epithelial repair, andmucosal architectural and glandular restoration. These data combinedwith the near normalization of MPO activity and marked reduction incytokine (ILa, ILb, IL6) expression indicate that therapy with the NACplus 5-ASA combination exerts a significantly greater anti-inflammatoryand reparative effect in TNBS colitis than either 5-ASA or NAC when usedalone.

Results in the DSS experiments demonstrated that DSS under theseexperimental conditions produced mild to moderate colitis. The aggregatemicroscopic injury score for DSS treated animals was 5.5±2.0; maximalscore 12. Monotherapy with either 5-ASA or NAC alone caused slightreduction in aggregate scores to 4.1±0.9 and 4.0±1.1, respectively.These values were not significantly different from DSS alone. Incontrast, combination therapy with the NAC plus 5-ASA combinationreduced global or aggregate histological injury score by 67% to 1.8±0.8.Furthermore, the NAC plus 5-ASA combination caused significantimprovement in epithelial damage when compared to DSS alone. DSS colitiswas associated with elevated CRP values of 7.0±0.6 mg/ml. CPR levelswere reduced substantially by concurrent therapy with NAC plus 5-ASA andvalues were 1.0±0.3 mg/ml. Similar reductions in CRP levels wereobserved with either 5-ASA or NAC alone. DSS treatment caused markedelevation in cytokine gene expression for IL 1a and IL 1b and thesevalues represented a 6.8 and 12.1 fold increase, respectively, in geneexpression of these cytokines when measured above control values.

NAC and 5-ASA, alone or in combination, substantially reducedDSS-induced IL1a and IL1b gene expression by 55-90% to levels thatapproximated control values. Conclusions from these data indicate thatintraluminal therapy with the NAC plus 5-ASA combination causedsignificant amelioration of mucosal injury induced by DSS. Combinationtreatment with the NAC plus 5-ASA combination results were associatedwith substantial reduction in serum CRP levels and proinflammatorycytokine gene expression.

Current considerations of the pathogenesis of mucosal inflammation inIBD involve a number of steps from antigen presentation and processingby macrophages to amplification of T cell activation and differentiationand cytokine production (1). In addition, inflammatory cells, includinggranulocytes and mononuclear cells, are recruited to the mucosa in ahighly coordinated fashion. Once present in the inflamed mucosa, tissueinjury is enhanced by neutrophil production of reactive oxygen speciessuch as superoxide and an increase in the expression of the inducibleisoform of NO synthase (iNOS) (5). Mucosal healing is thought to occur,in part, by reduction in injurious and proinflammatory substances and,also, by local liberation of growth factors which facilitate cellularrestitution and repair (2). Although several experimental approacheshave been employed that suggest the importance of enhanced production ofsuperoxide and nitric oxide in the pathogenesis of IBD, inconsistentresults have this issue unresolved (5). The beneficial effect superoxidedismutase (SOD) treatment in experimental models of colitis (21-23) hasshown limited effect in humans with IBD (24). In addition, iNOSinhibition has shown variable results in experimental models of IBD(25-27). However, recent studies using gene-targeted mice suggest adominant role of iNOS-derived NO in a murine model of dextran sulfatesodium (DSS) colitis (5). The antioxidants NAC and phenyl N-tertbutylnitrone have been shown to be effective in reducing the injuriousconsequences of TNBS colitis in rats (9) and DDS colitis in mice (10),respectively. Antioxidant therapy has also been demonstrated to suppresscolonic iNOS activity and to decrease NF-κB DNA-binding activity inexperimental colitis (10) and man (16).

In conclusion, our results showed that treatment of TNBS-induced colitiswith the NAC plus 5-ASA combination was superior to either 5-ASA or NACwhen used alone in reducing colonic inflammation and in promotingmucosal repair. In addition, combination therapy with the NAC plus 5-ASAcombination acted synergistically to result in, a significant reductionin MPO activity and proinflammatory cytokine gene expression.

Utility

The present invention provides a method for the treatment of a mammalsubject (including humans) afflicted with inflammatory diseases, and inparticular, inflammatory bowel diseases. The present invention hasseveral advantages over current therapies. As demonstrated herein,combination therapy with 5-ASA plus an antioxidant is synergisticallysuperior to either agent alone in controlling mucosal inflammation. Itis envisioned that such combination therapy would be capable of beingdelivered by oral route, employing existing coating technologies, tosites of inflammation. In addition, such combination therapy would beamenable to local therapy in the distal colon and rectum by enema orsuppository. Further description regarding deliver methods and dosingsystems and protocols is discussed below.

The combination of at least two active anti-inflammatory agents into asingle delivery system as described herein provides greater clinicalefficacy, development of a new pharmacochemical strategy for treatingmucosal inflammatory conditions, and a reduction in the need for otherpotentially toxic and expensive anti-inflammatory agents. The chemicalcoupling of 5-ASA to an antioxidant substance can provide a furtherpharmacological approach to the treatment of mucosal inflammatoryconditions such as IBD.

The 5-ASA used in the composition of the present invention may beprovided as the free acid, or as a pharmaceutically-acceptable salt orester, for example as described in U.S. Pat. No. 5,013,727, the entiretyof which is hereby expressly incorporated by reference herein.

Both 5-ASA and antioxidants such as NAC and phenyl N-tert-butylnitronehave relatively low profiles for toxicity. 5-ASA may be associated withallergic reactions to the medications and should be avoided in patientswith aspirin sensitivity. As with any nonsteroidal agent there existspotential for hepatic and renal toxicity. N-acetylcysteine or Mucomyst™has had a wide experience in man for the treatment of acetaminophenhepatotoxicity. This agent has proven safe. Other antioxidantscontemplated for use herein in conjunction with 5-ASA include otheraminosalicylates including 4-aminosalicylic acid (4-ASA), andN-acetyl-5-aminosalicylic acid; other nonsteroidal anti-inflammatorydrugs (NSAIDs) including those that inhibit cyclooxygenase I and/or II,such as sulindac, celecoxib and refacoxib; ascorbate; vitamin C; vitaminA; Vitamin E; beta-carotene; herbal agents such as milk thistle;selenium; iron in various ferric and ferrous formulations; phospholipaseA2 inhibitors, e.g., carboxymethylcellulose-linkedphosphatidylethanolamine; superoxide dismutase mimetics, such asMn(II/III) tetrakis (1-methyl-4-peridyl) of NmTMPyP; melatonin; zolimid;rebamipide; and phenyl N-tert-butylnitrone (PBN); and combinations ofany of the above.

While is it contemplated that applications of the invention would beprincipally of treating mucosal inflammatory conditions associated withinflammatory bowel diseases, such as ulcerative colitis, Crohn's diseaseand Behcet's disease, the invention would also have application to otherdisorders of the gastrointestinal tract such as radiation and infectiveenteritis, ischemic injury to the gastrointestinal tract, infectious,caustic agent-induced gastrointestinal injury, hemorrhagic rectal ulcer,ileum pouchitis, ischemic enteritis and drug-induced colitis, mucouscolitis, pseudomembranous enterocolitis, non-specific colonic ulcers,collagenous colitis, cathartic colon, ulcerative proctitis, idiopathicdiffuse ulcerative non-granulomatous enteritis, non-steroidalanti-inflammatory drug-induced inflammations, celiac sprue and the like.Furthermore, advances in our understanding of the pathogenesis ofgastrointestinal malignancies suggest a role for prostaglandinsgenerated by the cyclooxygenase enzymes to have a role in neoplasia. Ithas been well established that nonspecific and specific cyclooxygenaseinhibitors can reduce the propensity to neoplasia and malignancy inexperimental models and in human subjects. Full appreciation of the roleof prostaglandins in the cyclooxygenase system in the pathogenesis ofgastrointestinal malignancy is not complete. However, it is conceivablethat therapies which combine inhibition prostaglandin synthesis throughthe cyclooxygenase enzymes and antioxidant therapies may have abeneficial role in preventing gastrointestinal malignancies such ascolorectal cancer.

The term “inflammation” as used herein is meant to include reactions ofboth the specific and non-specific defense systems. A specific defensesystem reaction is a specific immune system reaction response to anantigen. Examples of a specific defense system reaction include theantibody response to antigens such as rubella virus, and delayed-typehypersensitivity response mediated by T-cells (as seen, for example, inindividuals who test “positive” in the Mantaux test).

A non-specific defense system reaction is an inflammatory responsemediated by leukocytes incapable of immunological memory. Such cellsinclude granulocytes, macrophages, neutrophils, for example. Examples ofa non-specific defense system reaction include the immediate swelling atthe site of a bee sting, the reddening and cellular infiltrate inducedat the site of a burn and the collection of PMN leukocytes at sites ofbacterial infection (e.g., pulmonary infiltrates in bacterialpneumonias, pus formation in abscesses).

Although the invention is particularly suitable for cases of acuteinflammation, it also has utility for chronic inflammation. Types ofinflammation that can be treated with the present invention includediffuse inflammation, traumatic inflammation, immunosuppression, toxicinflammation, specific inflammation, reactive inflammation,parenchymatous inflammation, obliterative inflammation, interstitialinflammation, croupous inflammation, and focal inflammation.

A therapeutically effective amount of a composition of the presentinvention refers to an amount which is effective in controlling,treating or moderating the inflammatory response. The terms“controlling”, “treating” or “moderating” are intended to refer to allprocesses wherein there may be a slowing, interrupting, arresting, orstopping of the progression of the disease and does not necessarilyindicate a total elimination of all disease symptoms.

The term “therapeutically effective amount” is further meant to definean amount resulting in the improvement of any parameters or clinicalsymptoms characteristic of the inflammatory response. The actual dosewill be different for the various specific molecules, and will vary withthe patient's overall condition, the seriousness of the symptoms, andcounter indications.

As used herein, the term “subject” or “patient” refers to a warm bloodedanimal such as a mammal which is afflicted with a particularinflammatory disease state. It is understood that guinea pigs, dogs,cats, rats, mice, horses, cattle, sheep, goats, pigs, llamas, and humansare among the examples of animals within the scope of the meaning of theterm.

A therapeutically effective amount of the compound used in the treatmentdescribed herein can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of conventionaltechniques and by observing results obtained under analogouscircumstances. In determining the therapeutically effective dose, anumber of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease or condition involved; the degreeof or involvement or the severity of the disease or condition; theresponse of the individual subject; the particular compoundadministered; the mode of administration; the bioavailabilitycharacteristic of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A therapeutically effective amount of the compositions of the presentinvention will generally contain sufficient active ingredient (i.e., theantioxidant and 5-ASA) to deliver from about 0.1 μg/kg to about 6000mg/kg (weight of active ingredient/body weight of patient). Preferably,the composition will deliver at least 1.0 μg/kg to 1000 mg/kg, and morepreferably at least 1 mg/kg to 100 mg/kg, although each dose of thecomposition may be more or less than these amounts. For example, thedaily dose for an adult may be in the range of about 10 mg to 300 mg/kg,preferably in the range of about 20 mg to 300 mg/kg, especially in therange of 50 mg/kg to 200 mg/kg. Also see U.S. Pat. No. 5,013,727 whichis incorporated by reference herein.

Practice of the method of the present invention comprises administeringto a subject a therapeutically effective amount of the compositiondescribed herein, in any suitable systemic or local formulation, in anamount effective to deliver the dosages listed above. The dosage can beadministered on a one-time basis, or (for example) from one to fivetimes per day or once or twice per week, or continuously via a venousdrip, depending on the desired therapeutic effect.

As noted, preferred amounts and modes of administration are able to bedetermined by one skilled in the art. One skilled in the art ofpreparing formulations can readily select the proper form and mode ofadministration depending upon the particular characteristics of thecompound selected, the disease state to be treated, the stage of thedisease, and other relevant circumstances using formulation technologyknown in the art, described, for example, in Remington's PharmaceuticalSciences, latest edition, Mack Publishing Co.

Pharmaceutical compositions can be manufactured utilizing techniquesknown in the art. Typically the therapeutically effective amount of thecompound will be admixed with a pharmaceutically acceptable carrier.

The compounds or compositions of the present invention may beadministered by a variety of routes, for example, orally, intrarectallyor parenterally (i.e., subcutaneously, intravenously, intramuscularly,intraperitoneally, or intratracheally).

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions, or emulsions. Solid unit dosage forms can becapsules of the ordinary gelatin type containing, for example,surfactants, lubricants and inert fillers such as lactose, sucrose, andcornstarch or they can be sustained release preparations.

In another embodiment, the compounds of this invention can be tablettedwith conventional tablet bases such as lactose, sucrose, and cornstarchin combination with binders, such as acacia, cornstarch, or gelatin,disintegrating agents such as potato starch or alginic acid, and alubricant such as stearic acid or magnesium stearate. Liquidpreparations are prepared by dissolving the active ingredient in anaqueous or non-aqueous pharmaceutically acceptable solvent which mayalso contain suspending agents, sweetening agents, flavoring agents, andpreservative agents as are known in the art.

For parenteral administration, the compounds may be dissolved in aphysiologically acceptable pharmaceutical carrier and administered aseither a solution or a suspension. Illustrative of suitablepharmaceutical carriers are water, saline, dextrose solutions, fructosesolutions, ethanol, or oils of animal, vegetative, or synthetic origin.The pharmaceutical carrier may also contain preservatives, and buffersas are known in the art.

The compounds of this invention can also be administered topically. Thiscan be accomplished by simply preparing a solution of the compound to beadministered, preferably using a solvent known to promote transdermalabsorption such as ethanol or dimethyl sulfoxide (DMSO) with or withoutother excipients. Preferably topical administration will be accomplishedusing a patch either of the reservoir and porous membrane type or of asolid matrix variety.

As noted above, the compositions can also include an appropriatecarrier. For topical use, any of the conventional excipients may beadded to formulate the active ingredients into a lotion, ointment,powder, cream, spray, or aerosol. For surgical implantation, the activeingredients may be combined with any of the well-known biodegradable andbioerodible carriers, such as polylactic acid and collagen formulations.Such materials may be in the form of solid implants, sutures, sponges,wound dressings, and the like. In any event, for local use of thematerials, the active ingredients usually be present in the carrier orexcipient in a weight ratio of from about 1:1000 to 1:20,000, but arenot limited to ratios within this range. Preparation of compositions forlocal use are detailed in Remington's Pharmaceutical Sciences, latestedition, (Mack Publishing).

Additional pharmaceutical methods may be employed to control theduration of action. Increased half-life and controlled releasepreparations may be achieved through the use of polymers to conjugate,complex with, or absorb the composition described herein. The controlleddelivery and/or increased half-life may be achieved by selectingappropriate macromolecules (for example, polysaccharides, polyesters,polyamino acids, homopolymers polyvinyl pyrrolidone,ethylenevinylacetate, methylcellulose, or carboxymethylcellulose, andacrylamides such as N-(2-hydroxypropyl) methacrylamide, and theappropriate concentration of macromolecules as well as the methods ofincorporation, in order to control release.

Another possible method useful in controlling the duration of action bycontrolled release preparations and half-life is incorporation of theglycosulfopeptide molecule or its functional derivatives into particlesof a polymeric material such as polyesters, polyamides, polyamino acids,hydrogels, poly(lactic acid), ethylene vinylacetate copolymers,copolymer micelles of, for example, PEG and poly(1-aspartamide).

Alternatively, it is possible to entrap the compostions in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization (for example, hydroxymethylcellulose orgelatine-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are disclosed inthe latest edition of Remington's Pharmaceutical Sciences.

U.S. Pat. No. 4,789,734 describe methods for encapsulating compositionsin liposomes and is hereby expressly incorporated by reference herein.Essentially, the material is dissolved in an aqueous solution, theappropriate phospholipids and lipids added, along with surfactants ifrequired, and the material dialyzed or sonicated, as necessary. A reviewof known methods is by G. Gregoriadis, Chapter 14. “Liposomes”, DrugCarriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the agents can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time, rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474;4,925,673; and 3,625,214 which are expressly incorporated by referenceherein.

When the composition is to be used as an injectable material, it can beformulated into a conventional injectable carrier. Suitable carriersinclude biocompatible and pharmaceutically acceptable phosphate bufferedsaline solutions, which are preferably isotonic.

For reconstitution of a lyophilized product in accordance with thisinvention, one may employ a sterile diluent, which may contain materialsgenerally recognized for approximating physiological conditions and/oras required by governmental regulation. In this respect, the sterilediluent may contain a buffering agent to obtain a physiologicallyacceptable pH, such as sodium chloride, saline, phosphate-bufferedsaline, and/or other substances which are physiologically acceptableand/or safe for use. In general, the material for intravenous injectionin humans should conform to regulations established by the Food and DrugAdministration, which are available to those in the field.

The pharmaceutical composition may also be in the form of an aqueoussolution containing many of the same substances as described above forthe reconstitution of a lyophilized product.

The compounds can also be administered as a pharmaceutically acceptableacid- or base-addition salt, formed by reaction with inorganic acidssuch as hydrochloric acid, hydrobromic acid, perchloric acid, nitricacid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organicacids such as formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, and fumaric acid, or by reaction with an inorganic basesuch as sodium hydroxide, ammonium hydroxide, potassium hydroxide, andorganic bases such as mono-, di-, trialkyl and aryl amines andsubstituted ethanolamines.

As mentioned above, the compounds of the invention may be incorporatedinto pharmaceutical preparations which may be used for therapeuticpurposes. However, the term “pharmaceutical preparation” is intended ina broader sense herein to include preparations containing a5-ASA/antioxidant composition in accordance with this invention, usednot only for therapeutic purposes but also for reagent or diagnosticpurposes as known in the art, or for tissue culture. The pharmaceuticalpreparation intended for therapeutic use should contain a“pharmaceutically acceptable” or “therapeutically effective amount” ofthe composition, i.e., that amount necessary for preventative orcurative health measures. If the pharmaceutical preparation is to beemployed as a reagent or diagnostic, then it should contain reagent ordiagnostic amounts of a 5-ASA/antioxidant combination.

All references, patents and patent applications cited herein are herebyincorporated herein in their entirety by reference.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings.

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1. A method of treating an inflammatory bowel disease in a mammal by reducing cytokine gene expression in colonic tissue of the mammal, the method comprising administering to the mammal by rectal delivery to a colon of the mammal a therapeutically-effective amount of 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and a therapeutically-effective amount of N-acetylcysteine or a pharmaceutically-acceptable salt thereof, whereby administration of the combination of 5-aminosalicylic acid, or a pharmaceutically-acceptable salt thereof, and N-acetylcysteine, or a pharmaceutically-acceptable salt thereof, causes reduced cytokine gene expression in colonic tissue of the mammal.
 2. The method of claim 1, wherein the cytokine is selected from the group consisting of IL 1a, IL 1b, IL-4, IL-6, and TNFα.
 3. The method of claim 1, wherein the 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and the N-acetylcysteine or a pharmaceutically-acceptable salt thereof, are together disposed in a pharmaceutically acceptable carrier.
 4. The method of claim 1, wherein the 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and the N-acetylcysteine or a pharmaceutically-acceptable salt thereof, are administered separately.
 5. The method of claim 1, wherein macroscopic injury or inflammation to colonic mucosa of the mammal is reduced.
 6. The method of claim 1, wherein microscopic injury to colonic mucosa of the mammal is reduced.
 7. The method of claim 1, wherein the mammal is a human.
 8. A method of treating an inflammatory bowel disease in a mammal by reducing myeloperoxidase activity in colonic tissue of the mammal, the method comprising administering to the mammal by rectal delivery to a colon of the mammal a therapeutically-effective amount of 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and a therapeutically-effective amount of N-acetylcysteine or a pharmaceutically-acceptable salt thereof, whereby administration of the combination of 5-aminosalicylic acid, or a pharmaceutically-acceptable salt thereof, and N-acetylcysteine, or a pharmaceutically-acceptable salt thereof, causes reduced myeloperoxidase activity in colonic tissue of the mammal.
 9. The method of claim 8, wherein the 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and the N-acetylcysteine or a pharmaceutically-acceptable salt thereof, are together disposed in a pharmaceutically acceptable carrier.
 10. The method of claim 8, wherein the 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and the N-acetylcysteine or a pharmaceutically-acceptable salt thereof, are administered separately.
 11. The method of claim 8, wherein macroscopic injury or inflammation to colonic mucosa of the mammal is reduced.
 12. A method of treating an inflammatory bowel disease in a mammal, comprising: contacting a colonic cell of the mammal with a therapeutically-effective amount of 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and a therapeutically-effective amount of N-acetylcysteine or a pharmaceutically-acceptable salt thereof.
 13. The method of claim 12, wherein macroscopic injury to colonic mucosa of the mammal is reduced.
 14. The method of claim 12, wherein inflammation in colonic mucosa of the mammal is reduced.
 15. A method of treating an inflammatory bowel disease in a mammal by reducing cytokine gene expression in colonic tissue of the mammal, the method comprising contacting a colonic cell of the mammal with a therapeutically-effective amount of 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and a therapeutically-effective amount of N-acetylcysteine or a pharmaceutically-acceptable salt thereof, whereby the combination of 5-aminosalicylic acid, or a pharmaceutically-acceptable salt thereof, and N-acetylcysteine, or a pharmaceutically-acceptable salt thereof, causes reduced cytokine gene expression in the cell.
 16. The method of claim 15, wherein the cytokine is selected from the group consisting of IL 1a, IL 1b, IL-4, IL-6, and TNFα.
 17. The method of claim 15, wherein macroscopic injury or inflammation to colonic mucosa of the mammal is reduced.
 18. A method of treating an inflammatory bowel disease in a mammal by reducing myeloperoxidase activity in colonic tissue of the mammal, the method comprising contacting a colonic cell of the mammal with a therapeutically-effective amount of 5-aminosalicylic acid or a pharmaceutically-acceptable salt thereof, and a therapeutically-effective amount of N-acetylcysteine or a pharmaceutically-acceptable salt thereof, whereby the combination of 5-aminosalicylic acid, or a pharmaceutically-acceptable salt thereof, and N-acetylcysteine, or a pharmaceutically-acceptable salt thereof, causes reduced myeloperoxidase activity in the cell.
 19. The method of claim 18, wherein macroscopic injury or inflammation to colonic mucosa of the mammal is reduced. 